Antioxidant Properties and Secondary Metabolites Profile of Hyptis colombiana at Various Phenological Stages

Hyptis colombiana (Lamiaceae family), a species also treated as Cantinoa colombiana in a recently segregated genus from Hyptis, is a perennial herb or subshrub native to the Andes of northern South America. H. colombiana leaves are commonly used in traditional medicine to treat respiratory and digestive illnesses. In this study, H. colombiana plants at different phenological stages (vegetative, flowering, and post-flowering) were harvested to obtain essential oils (EOs) and extracts (from fresh plant materials or post-distillation waste) whose chemical compositions and antioxidant activities were determined. H. colombiana EOs distilled by microwave-assisted hydrodistillation were analyzed by GC/MS/FID, and hydroalcoholic extracts obtained from fresh plant materials or post-distillation waste were analyzed by UHPLC-ESI+/−-Orbitrap-MS. The antioxidant activity was evaluated by the ABTS+• and ORAC assays. The principal compounds found in EOs were sesquiterpene hydrocarbons (65%); specifically, (E)-β-caryophyllene and germacrene D. Pyranone, rosmarinic acid, rutin, and p-hydroxybenzoic acid were the main constituents in H. colombiana extracts. After analyzing the chemical composition and antioxidant activity (ORAC) of EOs and hydroethanolic extracts from flowering H. colombiana plants, minimal variations were found. It is advisable to harvest H. colombiana plants during their flowering stage to acquire EOs and extracts that can be utilized in the agro-industry of EOs and their natural derivatives.


Introduction
In recent years, the trend of consuming natural ingredients in final products has increased. This has generated a rise in the demand for essential oils (EOs) and plant extracts as natural components in food [1][2][3]. Every year, in India, around six million tons of plant materials residue result from the distillation process of geranium (Pelargonium graveolens), lemongrass (Cymbopogon flexuosus), citronella (C. winterianus), palmarosa (C. martinii), and mint (Mentha arvensis) [4].
The distillation of aromatic and medicinal plants produces EO, hydrolate, and residual biomass. After their distillation, the plant materials are used for animal feed [5], composting [6], or biofuel [7]; however, these post-distillation residues may contain potentially useful bioactive molecules, such as phenolic compounds, with antioxidant [8] and antimicrobial [9] properties.
In 2022, the EO market was valued at USD 8.8 billion, having the highest contributions in the food, medicine, cleaning products, and aromatherapy sectors [10]. In Colombia, the import of EOs (USD 15.11 million, 2022) as input for industries was higher than the national production of EOs (USD 0.61 million, 2022) [11]. Table 1 shows the yields of EOs distilled by microwave-assisted hydrodistillation (MWHD) and the hydroalcoholic extracts obtained from fresh or post-distillation plant materials. The plant aerial parts were collected at the three phenological stages, i.e., vegetative, flowering, and post-flowering.
The yields of EOs and hydroalcoholic extracts obtained from dried plant materials of H. colombiana before and after its distillation were similar for plants collected at three phenological stages (Table S1). The yields of the EOs obtained were higher than those reported by Flores et al. [19] (0.4% (w/w)) for plants grown in Venezuela. This may be due to the different climatic and geographical conditions in which the plants grew.
The ethanolic extraction yields from aerial parts of fresh (undistilled) H. suaveolens reported by Mandal et al. [32] (2.64% w/w) and Medoatinsa [33] (8% w/w) were lower than those obtained from H. colombiana. No reports were found on hydroalcoholic extracts of H. colombiana plants before or after their distillation or on their variation within the phenological stages.

Analysis of EOs from H. colombiana Plants Collected at Different Phenological Stages
The chromatographic profiles of the EOs obtained by MWHD from H. colombiana plants harvested at three different phenological stages are displayed in Figure 1. Thirty-two compounds (relative amount >0.1%) found in these EOs are presented in Table 2 with their experimental linear retention indices (LRIs) and those found in the literature [34][35][36] for two chromatographic columns with polar and nonpolar stationary phases.     Table 2.  Table 2.
Twenty-eight terpenes, representing 90% of the EO compounds, were positively identified. The study of the fragmentation patterns, reflected in their mass spectra, allowed the tentative identification of two sesquiterpene hydrocarbons (Figures S1 and S2 in Supplementary Materials) and two oxygenated sesquiterpenes ( Figures S3 and S4). Table S2 shows the analytical figures of merit measured for the standard substances used for the quantification of compounds present in the H. colombiana EOs. The external standard calibration method was used to quantify the identified compounds with relative GC peak areas ≥ 2% in the chromatograms of H. colombiana EOs (Table S3). Table S5 shows the exact experimental masses, the measurement error (∆ppm < 3), and the product ions observed in the mass spectra of the identified compounds. Confirmatory identification was performed by comparing mass spectra and experimental retention times with those of standard compounds. The tentative identification was based on the study of the fragmentation patterns and data reported in the scientific literature. Fourteen compounds were identified in the hydroalcoholic extracts obtained before and after the distillation of H. colombiana aerial parts. These compounds include six flavones (apigenin-C,C-diglucoside, luteolin-C-hexoside-O-desoxyhexosyl, luteolin-7-Oglucoside, hydroxylated salvigenin, trihydroxy-trimethoxyflavone, and salvigenin), four hydroxycinnamic acids (p-hydroxybenzoic, caffeic, o-hydroxybenzoic, and rosmarinic acids), two flavonols (rutin and kaempferol-3-O-rutinoside), a sesquiterpene lactone, and a pyranone (Table S5).

Analysis of H. colombiana Hydroethanolic Extracts Using UHPLC-ESI +/− -Orbitrap-MS
A pyranone (compound N • 10,  Figure S6).  Table S5. Table S6 contains information on the quantification of the 14 compounds identified in the hydroalcoholic extracts obtained from H. colombiana plants before and after their distillation. The quantification parameters obtained from the external calibration method using standard compounds are reported in Table S2. Table 3 presents the antioxidant activity evaluation of the EOs and the hydroalcoholic extracts of H. colombiana and individual terpene and phenolic compounds identified in the H. colombiana EOs and extracts. These values were evaluated using the 2,2 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS +• ) and oxygen radical absorption capacity (ORAC) assays.

Chemical Composition of H. colombiana EOs
The EOs of H. colombiana contain mainly sesquiterpene and monoterpene hydrocarbons. The major compounds of the EOs of the plants collected at the vegetative, flowering, and post-flowering stages were sabinene, (E)-β-caryophyllene, germacrene D, and caryophyllene oxide. The concentrations of (E)-β-caryophyllene (230 mg substance/g of EO) and germacrene D (200 mg substance/g of EO) in the EOs distilled from plants gathered at vegetative, flowering, and post-flowering stages were not significantly different (Table S4); however, the highest variations were observed for sabinene in all EOs distilled from plants harvested at the three phenological stages (Table S4).
The main ingredient in the essential oils of H. colombiana is (E)-β-caryophyllene, a compound produced by many plants to protect themselves from plant-eating animals. This is achieved by attracting the creatures that naturally prey on those herbivores [48]. Inside the plant cells, (E)-β-caryophyllene is created in the cytosol by changing farnesyl diphosphate into a farnesyl carbocation, which then forms an 11-membered bicyclic hydrocarbon [49]. The United States Food and Drug Administration (FDA) has approved the use of (E)-βcaryophyllene as a food additive [1]. This ingredient is also utilized in the perfume industry to add a woody scent [50]. Researchers Dahham et al. [51] extracted (E)-β-caryophyllene from the essential oil of Aquilaria crassna Pierre ex Lecompte (Thymeleaceae) and found that it had strong antibacterial properties against Staphylococcus aureus (with a minimum inhibitory concentration of 3 µM), performing better than the reference antibiotic kanamycin (with a minimum inhibitory concentration of 8 µM). The compound also displayed activity against various fungi, including Trichoderma reesei, Penicillium citrinum, and Aspergillus niger [51]. Given that (E)-β-caryophyllene is the major component of the essential oil of H. colombiana, this plant could serve as an important source of this compound.

Chemical Composition of H. colombiana Extracts
A pyranone, rosmarinic acid, rutin, and p-hydroxybenzoic acid were the major compounds detected in the hydroalcoholic extracts obtained from H. colombiana plants before and after distillation (Table S6). Until now, no studies have been found on the chemical composition of hydroalcoholic extracts from H. colombiana plants. In the extracts of H. pectinata [24] and H. suaveolens [25,26], rutin and caffeic and rosmarinic acids were also found. Luteolin-O-glucoside, salvigenin, and kaempferol-3-O-rutinoside were also present in the H. colombiana extracts.
The The amounts of rosmarinic acid and rutin in hydroethanolic extracts from H. colombiana plants remained consistently steady, at 17 mg/g and 8 mg/g of dry extract, respectively (see Tables S6 and S7). This remained true regardless of the plant's growth stage or the method of distillation applied, even though distillation processes are usually associated with causing the breakdown of compounds in the remaining plant material.
Rosmarinic acid is present in plants of the Lamiaceae family, mainly in those of the Nepetoideae subfamily [53]. Rosmarinic acid is biosynthesized in plants from two amino acids, L-tyrosine and L-phenylalanine, through a reaction catalyzed by eight enzymes [54]. The function of rosmarinic acid in plants is to provide resistance to environmental stress and defense against pathogens and herbivores [55]. Furthermore, this compound has antimicrobial [56], antioxidant [57], anti-inflammatory [58], antimutagenic, and antiviral [54] properties.
Rosemary (Salvia rosmarinus Schleid) contains high levels of rosmarinic acid (24 mg/g) [59], which is approved by the European Union as a food preservative [60,61]. From every 100 g of H. colombiana dry plant material obtained before or after its distillation, up to 200 mg of rosmarinic acid could be isolated. This adds value to the residual plant material after its distillation because it can be used as a source of rosmarinic acid and as a possible food preservative.

Antioxidant Activity of H. colombiana EOs and Extracts
Many assays have been developed to evaluate the antioxidant capacity of vegetal extracts and other ingredients. Their reproducibility has been a frequent topic of discussion; after 25 years of use by many researchers for different applications, many recommendations have been formulated to obtain reliable results [62]. Kevers et al. performed the validation of the ORAC assay [63] and Xiao et al. published a detailed guideline for the execution of the most common assays [64].
The antioxidant activity values (µmol Trolox ® /g sample) of the EOs of H. colombiana evaluated by the ABTS +• assay presented significant differences (p > 0.05) for plants collected at the three phenological stages (Table S8). The highest values were obtained for EOs distilled from plants in the post-flowering stage (904 ± 2 µmol Trolox ® /g sample). It is possible that the antioxidant activity of the EOs is a result of a combination of various compounds, including caryophyllene oxide, sabinene, and (E)-β-caryophyllene. The individual antioxidant activities of these compounds were low, but together they may have a synergistic effect; however, the antioxidant activity values of the extracts obtained from plants collected at the three phenological stages did not show significant differences for the ORAC assay results (Table S8). The ORAC assay examines the antioxidant capacity of radical quenching mediated by the hydrogen transfer mechanism, while the ABTS assay also includes the electron transfer mechanism [65]. Therefore, the differences in antioxidant capacity of the EOs and extracts examined can be attributed to the differences in their content of substances that use electron transfer for interaction with radicals. The main compounds in the extracts of the H. colombiana plants collected at different vegetative stages (namely, a pyranone, rosmarinic acid, and rutin), did not have significant variations in their concentrations. H. colombiana hydroethanolic extracts exhibited lower antioxidant activity compared to the standard substances of rutin and rosmarinic acid. It is possible that these compounds are the primary contributors to the antioxidant activity found in the extracts. Other extract constituents that may contribute to the total antioxidant capacity are hydroxycinnamic acids and flavones.
The antioxidant activity of H. colombiana extracts has not been previously reported, but studies have been found on other species of the genus Hyptis spp. [65,66]. Tafurt

Distillation of Essential Oils
The aerial parts of the H. colombiana plants were harvested, dried in a shaded greenhouse, and chopped before their distillation. Plant material (100-350 g) and distilled water (200-350 mL) were added to a round-bottomed flask (2 L) placed inside a household microwave oven (Samsung (Suwon, Republic of Korea), model MS32J5133AG) with 1.6 kW output power and a radiation frequency of 2450 MHz. The total distillation time was 45 min, divided into three periods, each one lasting 15 min, with a 5 min rest time. Hydrodistillation was carried out in Clevenger-type equipment, with a Dean-Stark distillation reservoir with a ground joint to the round-bottomed flask. The EOs obtained were dried with anhydrous Na 2 SO 4 (J.T. Baker, Phillisburg, NJ, USA) before chromatographic analysis.

Solvent Extraction
Solvent extraction (SE) was performed as described by Medoatinsa et al. [33], with some modifications. To compare the yields and the chemical composition of the extracts, two SEs were carried out using plant materials before or after hydrodistillation, previously dried at room temperature. Dried and ground plant materials of H. colombiana (100 g) were mixed with an EtOH:H 2 O solution (2 L, 70:30 v/v) and deposited in an ultrasonic bath (Elmasonic S15H, Singen, Germany) at 50 • C, for 1 h. The mixture was vacuum filtered using a Büchner funnel (Whatman N • 1 filter paper), a Kitasato flask, and a vacuum pump (Vacuubrand, Wertheim, Germany). The extracts were rotoevaporated in the Heidolph equipment (Hei-VAP, Advantage HL, Chicago, IL, USA), then dried in a VirTis AdVantage Plus tray lyophilizer (SP Scientific, Gardiner, NY, USA) and stored at 4 • C, protected from light, before analysis. The quantification of the EO components was performed by an AT 6890N gas chromatograph (AT, Palo Alto, CA, USA), coupled to a flame ionization detector (FID). The separation of the oil components was carried out in a capillary column with the apolar stationary phase (DB-5MS) of the same dimensions as the one used for GC/MS analysis. Samples were injected in split mode (1:30), the injector temperature was 250 • C, the injection volume was 1 µL, and the FID temperature was maintained at 280 • C. Quantification was performed using calibration curves of the reference substances of α-pinene, sabinene, pcymene, limonene, γ-terpinene, linalool, (E)-β-caryophyllene, germacrene D, α-humulene, and caryophyllene oxide, among others. The data were processed with GC ChemStation software version B.04.03-SP1 (2001-2012).

LC/MS Extract Analysis
The analysis was carried out in an ultrahigh performance liquid chromatograph, UHPLC Vanquish (Thermo Fisher Scientific, Germering, Germany), equipped with a Q-Exactive Plus Orbitrap mass detector (Thermo Scientific, Bremen, Germany), an electrospray interface with heating (HESI-II), operated in dual acquisition mode of positive and negative ions, at 350 • C, a degasser (SRD-3400), a binary pump (HPG-3400RS), an autosampler (WPS-300TRS), and a thermostated unit (TCC-3000) to house the guard and the analytical columns. Separation of the components of the mixture was performed on a ZORBAX Eclipse XDB-C 18 column (Sigma Aldrich, St. Louis, MO, USA), 50 mm, L × 2.1 mm, i.d., × 1.8 µm particle size, at 30 • C. The mobile phase was: A-water (0.1% formic acid + 5 mM HCOONH 4 ) and B-methanol (0.1% formic acid + 5 mM HCOONH 4 ). Nitrogen (>99%) was supplied by an NM32LA generator (Peak Scientific, Inchinnan, Scotland, UK) and used as both the drying and nebulizing gas. The capillary temperature was 320 • C, and the voltage was 3.5 kV. The ions passed through a quadrupole, a C-Trap ion trap, and a higher energy dissociation collision cell (HCD). Values of 10, 20, 30, and 40 eV were used in the HCD. Data were processed with Thermo Xcalibur TM Roadmap software, Version 3.1.66.10. The identification of compounds was carried out based on the extracted ion current (EIC) of protonated [M+H] + or deprotonated [M-H] − molecules, depending on the case, and by comparison with the mass spectra of substances in databases (HMDB [68] and MassBank [69]) and reference substances (Section 4.1).

Decoloration of the ABTS +• Cation-Radical Assay
The ABTS +• cation-radical decoloration assay was carried out as described by Re et al. [70], with some modifications [71]. The test was performed on a Varioskan LUX VL0000D0 microplate reader (Thermo Scientific, Singapore) using transparent 96-well microplates. ABTS (7 mM in sodium acetate buffer, pH 4.5) and potassium persulfate (2.45 mM) were mixed with sonication for 30 min; the mixture was stored at 4 • C (24 h) in the absence of light to obtain a stable ABTS +• solution. ABTS +• was diluted in acetate buffer until an absorbance of 0.71 ± 0.02 at λ = 750 nm was obtained; the mixture was stored at 4 • C (30 min) before use. The EOs and extracts, obtained from plant materials before and after hydrodistillation, were weighed (10 mg), dissolved in methanol, and diluted in acetate buffer (20 mM, pH 4.5). The EO or the diluted extract and the ABTS +• solution were deposited in each well of the plate and the absorbance was measured for 60 min. Trolox ® (97%, Sigma-Aldrich, St. Louis, MO, USA) was used as the reference substance. Three independent experiments were carried out (n = 3) and the results were expressed as the mean value ± standard deviation of µmol Trolox ® /g of EO or dry extract.

Evaluation of the Oxygen Radical Absorption Capacity
The oxygen radical absorbance capacity was determined, according to Ou et al. [72], with some modifications [71]. Measurements were performed using a Varioskan LUX VL0000D0 microplate reader (Thermo Scientific, Singapore) equipped with black 200 µL 96-well poly(styrene) microplates, under fluorescence mode. The EOs or extracts were weighed (1 mg) and dissolved in methanol, and then dilutions were made in phosphate buffer; in each well of the plate, the EO or the diluted extract (25 µL) and the fluorescein solution (150 µL, 81 nM in phosphate buffer) were deposited; the mixture was incubated at 37 • C (20 min), and a solution of AAPH (25 µL, 153 mM, in phosphate buffer) was added. Fluorescence was measured (37 • C, 90 min) with an excitation wavelength of λ = 490 nm and an emission wavelength of λ = 510 nm. The antioxidant protection was determined from the difference between the area under the curve (AUC) obtained for each sample and for the blank (25 µL of phosphate buffer). Trolox ® (97%, Sigma-Aldrich, St. Louis, MO, USA) was used as the reference substance. Three replicates were carried out (n = 3) and the results were expressed as the mean value ± standard deviation of µmol Trolox ® /g of EO or dry extract.

Data Analysis
Analysis of variance (ANOVA) was used to determine significant differences (p < 0.05) between yields, chemical compositions, and antioxidant activities of H. colombiana essential oils and hydroethanolic extracts. ANOVA and Tukey's test were performed in InfoStat software (Version 2018).

Conclusions
Thirty-two compounds (relative amount > 0.1%) were found in EOs distilled from H. colombiana plants collected at the three phenological stages (vegetative, flowering, and postflowering); (E)-β-caryophyllene, germacrene D, sabinene, and caryophyllene oxide were the most abundant components. Fourteen compounds were identified in the hydroalcoholic extracts obtained from plant material before or after distillation: a pyranone (70 mg/g), rosmarinic acid (17 mg/g), and rutin (8 mg/g) were the major components. The chemical composition of the EOs and extracts of H. colombiana, obtained from plants collected at the three phenological stages, did not present appreciable differences in the amounts of their major compounds. The phenological stage of the plant had little effect on the amounts of the main secondary metabolites in the EOs or extracts. The antioxidant activity values obtained from H. colombiana extracts showed little variation when the plants were collected in bloom at different harvests. The flowering phenological stage could be considered propitious for the collection of plant materials to obtain extracts with the highest antioxidant activity. The native plant H. colombiana, promising for its bioactive compounds, could be easily cultivated in Cundinamarca, Boyacá, Santander, and other regions of Colombia with medium altitudes, where this species has been found growing in poor soils between 600 and 2700 m above sea level [15]. Thus, it could integrate the production chain of natural ingredients for different bioproducts under circular economy schemes that employ the residual biomass from distillation to obtain extracts and individual bioactive compounds of interest to cosmetic and pharmaceutical industries.   Table  S5. Figure